Perennial ryegrass (Lolium perenne L.) is the most widely cultivated forage, turf and amenity grass species of global temperate grazing zones. Favourable agronomic qualities include high dry matter yield, nutritive content, digestibility, palatability and the ability to recover from heavy defoliation by herbivores [1, 2]. Perennial ryegrass is, however, susceptible to a number of different foliar diseases. Crown rust (Puccinia coronata f.sp. lolii) is the most widespread and damaging disease affecting ryegrasses [3–7]. Stem rust (P. graminis f.sp. lolii) infections are especially serious for producers of ryegrass seed , while grey leaf spot (Magnaporthe grisea), dollar spot (Sclerotinia homoeocarpa) and brown patch (Rhizoctonia solani) reduce turf quality . The development of cultivars resistant to each of these diseases is currently recognised as an important mode of infection control.
The obligate outbreeding reproductive habit of perennial ryegrass  leads to high levels of genetic variation within, and to a lesser extent, between cultivars [11–13]. Conventional breeding for disease resistance is hence anticipated to be relatively slow for outcrossing forage species as compared to allogamous species such as cereals, because of a requirement for extensive progeny screening and phenotyping. Nonetheless, major genes and quantitative trait loci (QTLs) for disease resistance have been detected in Lolium species for resistance to crown rust [14–21], stem rust , bacterial wilt , powdery mildew  and grey leaf spot . The extent of genetic variation within temperate Australasian crown rust pathogen populations  is consistent with the presence of different virulence races . Identification of the molecular basis of major resistance determinants to different pathotypes will improve selection of favourable alleles during cultivar development.
Both genetic and physiological analysis has determined that hypersensitive reactions in response to fungal, viral and bacterial pathogen infections are caused by the action of genes encoding receptor proteins [28, 29]. The major class of resistance (R) genes contain a highly conserved nucleotide binding site (NBS) domain adjacent to the N-terminus and a leucine-rich repeat (LRR) domain involved in the host recognition of pathogen-derived elicitors. NBS-LRRs constitute one of the largest plant gene families, accounting for c. 1% of all open reading frames (ORFs) in both rice and Arabidopsis thaliana, and are distributed non-randomly throughout the genome [30–32]. Clustering of R genes is known to facilitate tandem duplication of paralogous sequences and generation of new resistance specificities to counter novel avirulence determinants in evolving pathogen populations [30–34].
NBS domain-containing sequences have been isolated using degenerate PCR from many agronomically-important Poaceae species including cereals [33–37] and forage grasses [24, 38, 39]. In a comparison with the fully-sequenced rice genome , only a small proportion of the total NBS domain sequences are so far likely to have been isolated from Lolium species. Multiple strategies are hence required to isolate a larger R gene sample, allowing for structural characterisation, marker development for genetic mapping, and the potential for correlation with the locations of known disease resistance loci.
Disease resistance loci of cereal species are conserved at the chromosomal and molecular level [40, 41], and provide valuable template genes for a translational genomic approach to molecular marker development . For example, the TaLrk10 receptor kinase gene (located at the Lr10 locus on hexaploid wheat chromosome 1AS) has been found to confer resistance to leaf rust in specific cultivars, and putative Lrk10 ortholoci are structurally conserved between Poaceae species [41, 43]. The Lrk10 orthologue of cultivated oat (Avena sativa L.) exhibits 76% nucleotide similarity to the wheat gene and maps in a region of conserved synteny between the two genomes, co-locating with a large cluster of NBS-LRR genes conferring resistance to the oat form of crown rust (P. coronata f.sp. avenae) . The Poaceae sub-family Pooideae includes perennial ryegrass, along with cereals of the Aveneae and Triticeae tribes [44, 45], suggesting that template genes from these species are highly suitable for ortholocus isolation.
Based on studies of cereal-pathogen interactions, similar qualitative and quantitative genetic mechanisms are likely to contribute to disease resistance in perennial ryegrass. In order to test this hypothesis, a broad survey based on empirical and computational approaches was conducted to recover an enhanced proportion of perennial ryegrass NBS domain-containing sequences, as well as specific R gene ortholoci. Candidate R gene sequences (referred to as R genes throughout the text) were characterised by functional annotation, motif structure classification and phylogenetic analysis. Single nucleotide polymorphisms (SNPs) were discovered through re-sequencing of haplotypes from the parents of a two-way pseudo-testcross mapping population and validated SNPs were assigned to genetic maps. Co-location with disease resistance QTLs was demonstrated within Lolium taxa and by comparative analysis with related Poaceae species.